Stephen J. Fuller

The P2X7 receptor (or P2X7) is an ATP-gated ion channel with important roles in the immune and other body systems. Much has been garnered about P2X7 using the first published anti-human P2X7 monoclonal antibody (mAb) (clone L4). This mAb can be used to impair P2X7 channel and pore activity and events downstream of P2X7 activation in vitro and in vivo. Immunolabelling with this mAb has been used in flow and mass cytometry, confocal microscopy and immunohistochemistry to help establish the presence of P2X7 on human leukocytes and other primary cell types, as well as various malignant cell lines. This mAb has been applied to immunoprecipitate P2X7 to identify co-associated molecules to help uncover novel roles of the receptor such as phagocytosis. Recently, this mAb has been shown to mediate complement-dependent cytotoxicity, offering a new avenue of limiting P2X7-mediated processes by depleting P2X7 ⁺ cells. Finally, this mAb has been used to identify and characterise polymorphic and other variants of P2X7. This article will discuss the origins and various applications of the anti-P2X7 mAb (clone L4). As an early user of this mAb and pioneer of other purinergic technologies, this article is a tribute to the late Francesco Di Virgilio.

The P2X7 receptor (P2X7R) is a ligand-gated, non-selective cation channel activated by extracellular ATP, a key danger signal in the cellular stress response. Due to its roles in inflammation and neurological disorders, it is an attractive therapeutic target. However, clinical trials of P2X7R antagonists have failed to show clinical efficacy. This review explores whether receptor polymorphisms, alternative splicing, and membrane composition contribute to these clinical trial failures. Genotyping of trial participants is highly recommended prior to enrolment, and in vitro functional studies should be wary of the membrane composition of cells expressing P2X7R. While the P2X7R shows promising therapeutic potential, there remain large gaps in research particularly in characterising haplotypes and alternatively spliced hetero- or homotrimers of the receptor. This results in the development and testing of agents without considering the genetic variability of the receptor, which we propose to be a large contributor to the lack of clinical success. We also summarise characteristics of the receptor and recent structural findings to discuss how computational approaches may help overcome these challenges of variability. Precision targeting of the receptor in disease states is warranted, and a collaborative approach covering multiple facets of the receptor will facilitate this.

Prediabetes, characterized by impaired fasting glucose and/or glucose tolerance, is associated with organ damage, increased mortality, and accelerated aging, even before diabetes onset. Severe short‐term energy restriction while maintaining essential nutrient intake is among the most effective strategies for weight loss, metabolic health improvement, and delaying type 2 diabetes progression. Extracellular vesicles contribute to these metabolic benefits; however, the impact of energy‐restriction‐induced weight loss on the extracellular vesicle proteome remains incompletely understood. This study employed targeted and untargeted proteomics to investigate the effect of an 8‐week severely energy‐restricted diet on the plasma proteome in adults with prediabetes from Sydney, Australia, as part of the PREVIEW study. Circulating extracellular vesicles were enriched in plasma using an immunoaffinity‐based protocol. A total of 44 participants who achieved at least a 12% weight loss and provided informed consent were included in the study. Paired changes in over 2000 proteins between baseline and week 8 were analyzed. Following the intervention, multiple proteins associated with inflammation and senescence were significantly reduced, reversing the increase commonly associated with aging. The decline in inflammatory and senescence markers may have been mediated by extracellular vesicles, as indicated by significantly lower circulating levels of several vesicular markers. Additionally, several markers of protein synthesis downstream of mTORC1 and protein degradation were significantly reduced in vesicle‐enriched plasma, suggesting decreased intercellular secretion and/or trafficking. Overall, this study identifies a diet‐induced proteomic signature suggestive of reduced inflammation, lower senescence, and enhanced vesicle‐associated proteostasis, potentially conferring health benefits beyond glycemic control.

Hypomethylating agents are frontline therapies for myelodysplastic neoplasms (MDS), yet clinical responses remain unpredictable. We conducted a phase 2 trial comparing injectable and oral azacitidine (AZA) administered over one or three weeks per four-week cycle, with the primary objective of investigating whether response is linked to in vivo drug incorporation or DNA hypomethylation. Our findings show that injection results in higher drug incorporation, but lower DNA demethylation per cycle, while global DNA methylation levels in mononuclear cells are comparable between responders and non-responders. However, hematopoietic stem and progenitor cells (HSPCs) from responders exhibit distinct baseline and early treatment-induced CpG methylation changes at regulatory regions linked to tissue patterning, cell migration, and myeloid differentiation. By cycle six—when clinical responses typically emerge—further differential hypomethylation in responder HSPCs suggests marrow adaptation as a driver of improved hematopoiesis. These findings indicate that intrinsic baseline and early drug-induced epigenetic differences in HSPCs may underlie the variable clinical response to AZA in MDS.

B cells are central to the adaptive immune response and provide long-lasting immunity after infection. B cell activation is mediated by the surface membrane-bound B cell receptor (BCR) following recognition of a specific antigen. The BCR has been challenging to analyse using mass spectrometry (MS) due to the difficulty of isolating and enriching this membrane-bound protein complex. There are approximately 120,000 BCRs on the B cell surface; however, depending on the B cell activation state, there may be hundreds-of-millions to billions of proteins in a B cell. Consequently, advanced proteomic techniques such as MS workflows that use purified proteins to yield structural and protein-interaction information have not been published for the BCR complex. This paper describes a method for enriching the BCR complex that is MS-compatible. The method involves a Protein G pull down on agarose beads using an intermediary antibody to each of the BCR complex subcomponents (CD79a, CD79b, and membrane immunoglobulin). The enrichment process is shown to pull down the entire BCR complex and has the advantage of being readily compatible with further proteomic study including MS analysis. Using intermediary antibodies has the potential to enrich all isotypes of the BCR, unlike previous methods described in the literature that use protein G-coated beads to directly pull down the membrane IgG (mIgG) but cannot be used for other mIg isotypes.

Objectives: To examine the relationship between vitamin C status and demographic factors in New South Wales on the basis of serum vitamin C test results undertaken at the central pathology laboratory in Sydney, and to assess associations with age, gender, social disadvantage, and geographic remoteness.Design, setting: Retrospective observational study; analysis of vitamin C test results undertaken at the Royal Prince Alfred Hospital, 1 January 2017 – 31 December 2021.Main outcome measures: Vitamin C status (normal, serum concentration ≥ 40 μmol/L; hypovitaminosis C, 12–39 μmol/L; significant deficiency, < 12 μmol/L); associations of vitamin C status with year of testing, age, gender, socio‐economic status (Index of Relative Socio‐Economic Advantage and Disadvantage quintile), and geographic remoteness (Australian Statistical Geography Standard); rate of hypovitaminosis C or significant deficiency test results (relative to findings of normal levels; per 100 000 estimated resident population) by Statistical Area 3.Results: Of 17 507 vitamin C tests undertaken during 2017–2021, 4573 were excluded (multiple tests for individuals); of 12 934 included results, 6654 were for women (51.5%), 9402 for people living in major cities (73.5%), and 81 for people in remote or very remote areas (0.6%). In multivariable multinomial regression analyses, significant deficiency (relative to normal test results) was more likely for men than women (adjusted odds ratio [aOR], 1.39; 95% confidence interval [CI], 1.27–1.52); the likelihood of hypovitaminosis C (IRSAD quintile 1 v 5, aOR, 1.35; 95% CI, 1.19–1.53) or significant deficiency (aOR, 2.07; 95% CI, 1.79–2.40) generally increased with postcode‐level socio‐economic disadvantage. Several of the population areas with the highest low vitamin C rates were areas of greatest disadvantage in NSW.Conclusions: The prevalence of vitamin C deficiency among older people and people living in areas of socio‐economic disadvantage indicates that population assessment of vitamin C levels would be appropriate.

B cells are central to the adaptive immune response, providing long lasting immunity after infection. B cell activation is mediated by a cell surface B cell receptor (BCR) following recognition of an antigen. BCR signaling is modulated by several co-receptors including CD22 and a complex that contains CD19 and CD81. Aberrant signaling through the BCR and co-receptors promotes the pathogenesis of several B cell malignancies and autoimmune diseases. Treatment of these diseases has been revolutionized by the development of monoclonal antibodies that bind to B cell surface antigens, including the BCR and its co-receptors. However, malignant B cells can escape targeting by several mechanisms and until recently, rational design of antibodies has been limited by the lack of high-resolution structures of the BCR and its co-receptors. Herein we review recently determined cryo-electron microscopy (cryo-EM) and crystal structures of the BCR, CD22, CD19 and CD81 molecules. These structures provide further understanding of the mechanisms of current antibody therapies and provide scaffolds for development of engineered antibodies for treatment of B cell malignancies and autoimmune diseases.

The P2X7 receptor is a trimeric ligand-gated cation channel activated by extracellular adenosine 5′-triphosphate. The study of animals has greatly advanced the investigation of P2X7 and helped to establish the numerous physiological and pathophysiological roles of this receptor in human health and disease. Following a short overview of the P2X7 distribution, roles and functional properties, this article discusses how animal models have contributed to the generation of P2X7-specific antibodies and nanobodies (including biologics), recombinant receptors and radioligands to study P2X7 as well as to the pharmacokinetic testing of P2X7 antagonists. This article then outlines how mouse and rat models have been used to study P2X7. These sections include discussions on preclinical disease models, polymorphic P2X7 variants, P2X7 knockout mice (including bone marrow chimeras and conditional knockouts), P2X7 reporter mice, humanized P2X7 mice and P2X7 knockout rats. Finally, this article reviews the limited number of studies involving guinea pigs, rabbits, monkeys (rhesus macaques), dogs, cats, zebrafish, and other fish species (seabream, ayu sweetfish, rainbow trout and Japanese flounder) to study P2X7.